1. Field of the Invention
This invention relates to a method, an apparatus, and kit for performing purification of nucleic acids, proteins and cells. More specifically, the invention relates to an apparatus and methods for purification and concentration of nucleic acids, proteins (e.g., antigens and antibodies) and cells without the need of centrifugation, precipitation or lengthy incubations. The apparatus and methods can be adapted to non-specific or specific capture of nucleic acids, proteins or cells in a biological or environmental samples and can be adapted for detection of the captured moiety by enzymatic calorimetric, fluorescent, luminescent or electrochemical formats with or without nucleic acids amplification.
2. Description of Related Art
Nucleic acids preparation and purification is essential to virtually all molecular biology. Most methods in use for purifying nucleic acids rely on labor-intensive organic extractions and/or centrifugation. In recent years, a new class of analytical and purification techniques have been developed which rely on the inherent biological affinities between proteins, between enzymes and their substrates, and between proteins and nucleic acids.
Affinity techniques are attractive because the desired molecules are rapidly and specifically immobilized away from the other contaminating molecules in an impure mixture, offering rapid and extensive purification or enrichment levels. Contaminating molecules are simply washed away, while target molecules remain firmly affinity-bound. Target molecules may be detached from their counterpart molecules simply by altering the environment to disfavor the affinity between the two.
In one technique, a solid phase support is used to attach target molecules from a sample, such as DNA, RNA, proteins or cells. The solid phase support can also be coated with specific oligonucleotides, peptide or cell receptors to capture a specific DNA, RNA or protein molecules as well as whole cells or microorganisms. Such solid phase supports consist generally of material with selective adsorption, ion exchange and catalytic properties. When such solid phase supports are formed by deep reactive ion etching (DRIE), they can provide exceptionally large surface area, high levels of activity and selectivity in a wide range of reactions, for example to nonspecifically capture electrically charged molecules, or specifically capture molecules through affinity binding. Examples of solid phase supports include silica-based material, synthetic polymers and a host of other naturally-occurring or chemically modified elements.
Chemical modification may be achieved by incorporating metal atoms, e.g., Li, Be, Mg, Co, Fe, Mn, Zn, B, Ga, Fe, Ge, Ti, Au, Pt or As into a solid support framework consisting of, for example, Si4+ and Al3+. In a typical application of a solid support system to directly capture nucleic acids molecules, for example, is to mix a biological sample with a guanidine-based lysis/binding solution in the reservoir, the sample capture assembly is inserted into the reservoir, sealed, the entire apparatus is briefly vortexed, agitated or sonicated, briefly incubated at the appropriate temperature, e.g., 37° C. (the shaft may also be thermally regulated through an attachment to a miniaturized thermal regulator) to allow the released nucleic acids to adsorb or bind to the capture assembly. Mechanical disruption (by vortexing, sonication or shaking) or enzymatic disruption (e.g., by lysozymes, proteinase K, collagenase) may be required for some biological samples to enhance the release of nucleic acids.
After the nucleic acids are released and captured onto the capture assembly by virtue of electrical charge or affinity binding, the capture assembly is removed, placed into another reservoir containing wash buffer with appropriate salt concentration and ionic strength (e.g., 1.0 M NaCl, 50 mM MOPS, 15% ethanol, pH 7.0 for DNA), sealed and briefly vortexed or agitated. Several washes can be performed in the same reservoir by replenishing the wash buffer if multiple washing is necessary to remove undesirable or inhibitory material from the captures nucleic acids. The removal of undesirable or inhibitory material can enhance subsequent nucleic acids amplification steps.
After washing, the reservoir is replaced with a fresh reservoir containing elution buffer with appropriate salt concentration and ionic strength (e.g., 1.25 M NaCl, 50 mM Tris/HCl, 15% ethanol, pH 8.5 for DNA), and the capture assembly is inserted into the reservoir, incubated at the appropriate temperature, e.g., 65° C. for several minutes (or the capture assembly is subjected to the appropriate elution temperature through the thermal regulator attachment). Alternatively, it is possible to perform thermal cycling through the thermal regulator attachment while the DNA is initially bound to the capture assembly with the appropriate nucleic acids amplification buffer and reagents placed in the reservoir. The Lysis/binding, washing and elution buffer conditions may be adapted according to the sample type and the type of the nucleic acids (DNA or RNA).
However, the solid phase supports currently available do not provide vast surface area to maximize binding of molecules. In addition, they are expensive to make, and do not lend themselves to in-home or field use because of either their size or configuration. Furthermore, they do not allow the flexibility of purifying different types of molecules, e.g., nucleic acids, proteins or whole cells in a single format with the ability to capture such molecules specifically or nonspecifically, and detect such molecules (specially nucleic acids) with or without nucleic acids amplification using colorimetric, fluorescent, luminescent or electrochemical formats. The present invention, in toto, allows much greater flexibility and efficiency and is adaptable to future modification by, for example, incorporating thermal cycling amplification (e.g. PCR), isothermal amplification and fluorogenic, calorimetric, luminescence or electrochemical detection in the same device. The present invention also allows incorporation of specific capture molecules, e.g. dendritic (branched) oligonucleotides or peptides to further increase the capture surface area and allow the specific capture of nucleic acids, cells or proteins. In addition, the invention can be adapted to an arrayable platform to allow high throughput sample processing and detection in the same device.
What is lacking in the art is a simple, inexpensive apparatus, flexible kit and method for DNA, RNA, protein, antigen, antibody or cell purification that can be used in the field, home or laboratory with the flexibility described above. In particular, what is needed is an apparatus and method that does not require centrifugation, precipitation, lengthy incubations, or extensive equipment and that provides a massive surface area for maximum exposure to and binding of target molecules. With an increasing desire to perform rapid testing for a variety of infectious disease agents or biological markers in the home, field or by medical and health care workers, there is a need to provide a simple, flexible and easy to use apparatus, kit and methods for purification and detection.
It is therefore an object of the invention to provide a simple method, apparatus and kit for conducting DNA, RNA, protein or cell purification.
It is a further object of the invention to provide an apparatus that is convenient to use in the home or in the field that provides high precision and good economy.
It is still a further object of the invention to provide an apparatus that provides efficient purification of nucleic acids, proteins, or cells with out the need for centrifugation, precipitation or lengthy incubations.
It is a further object of this invention to provide an apparatus that is adaptable for direct detection of nucleic acids and proteins by calorimetric, fluorogenic, luminescence or electronic means or detection of nucleic acids molecules after nucleic acids amplification in such an apparatus.
It is a further object of this invention to provide an apparatus and concept that is a adaptable for rapid, flexible high through put screening of biological samples or biological products for infectious disease agents and biomarkers.
These and other objects are achieved with the method, apparatus and kit of the present invention.